The present invention relates to the preparation of membranes and their use in effecting enzymatic and biological reactions and separations.
The present invention encompasses a group of matrix membranes, coupling reactions, proteins, enzymes and antibodies, in some respects similar to those of U.S. Pat. Nos. 4,0333,817, 4,033,822 and 4,863,714. As there, there are provided membrane filters to which enzymes and other molecules of similar catalytic or binding activity are attached via chemical bonds, where activation of the pore surfaces of these membranes or filters for purposes of subsequent coupling (where such is needed) can be carried out under pressure-driven conditions, and where the resulting coupled enzyme or biopolymer system can be used under pressure-driven conditions, i.e., by forcing the substrate to be treated through the membrane pores under pressure, to effect useful conversions and separations.
The conventional uses of immobilized biopolymers are well-known in the scientific, industrial and patent literature. The advantages of the immobilization procedure are well-known. The technologies which are usually employed have involved the use of fine particles of either natural or synthetic materials, often of organic polymers or porous materials of other substances, to which enzymes, proteins and other biopolymers are coupled by chemical bonds. These conventional processes have advantages and various disadvantages.
Further, membranes composed of natural polymers such as cellulose or of proteins have been employed, but these are susceptible to degradation or attack by microorganisms and enzymes. A number of purely synthetic membranes have been employed as carriers for protein immobilization, but these are usually not porous membranes but rather in the form of sheets coated with coupling groups, or systems where the active agent such as an enzyme fills the pores and process rates are diffusion-controlled, in large measure.
Still further, polymers of acrylonitrile (PAN) have seldom found use for enzyme immobilization. Where they have, it was under conditions which presented many problems of a preparative nature or in use. For example, proteins have been ionically bound to an acrylonitrile homopolymer after partial derivatization of the nitrile groups to imidoesters; however, only a small amount of protein could be bound. Similarly, amine monomers can be grafted onto PAN polymerized in the presence of bromoform, e.g., the photografting of N,N-dimethylaminoethyl methacrylate, followed by quaternization of the amine and then used via the process of ionic immobilization for the enzyme urease. Further, one can introduce amines into PAN by partial reduction of the nitrile groups; this facilitates the adsorption of proteins which can then be subsequently cross-linked with gluteraldehyde into a stable network. Such supports, however, have limited utility because the activation/coupling chemistries are not versatile and the process results in weak chemical bonds which cannot prevent enzyme leakage due to solvolytic processes.
It is accordingly an object of the present invention to provide ways of immobilizing biopolymers so that they can still engage in biological reactions and interactions to substantially the same extent as the mobile biopolymers prior to immobilization.
It is a further object of the invention to provide a way of producing from acrylonitrile polymers which in membrane form have high versatility and utility.
These and other objects and advantages are realized in accordance with the present invention pursuant to which there are provided novel methods of preparing such membranes, improvements in the coupling chemistries which are employed, and the use of these systems for specific applications of value. Where the system is used as a catalyst for the carrying out of a specific chemical reaction such as the isomerization of glucose, in the manner as described hereinbelow, the system displays a high capacity in terms of its enzyme content and, accordingly, a high enzymatic activity, making this system useful for the large-scale industrial process of glucose isomerization. Enzymes so stabilized have, in addition to the above-mentioned advantages, an intrinsic advantage in terms of a high chemical and thermal stability.
Similarly, when to these membrane filters or ultrafilters are coupled specific ligands, namely substances capable of forming specific complexes with certain species present in a mixture with other substances which may be similar in nature, where the specific agents and ligands are attached by chemical bonds to the inner pore surface of the membrane under pressure-driven conditions, then a process termed affinity sorption, as described in U.S. Pat. No. 4,163,714 can take place, similar to the well known process of affinity chromatography, but possessing unique advantages of speed and convenience. In affinity sorption, the pore diameters and chemical nature of these membranes must be such as to allow for the coupling of a high concentration of ligand, and also to provide for the ready access of the solute molecules whose separation and purification via reversible binding to the ligand substances is desired. The nature of these membranes is such that the excess, undesired components of the mixture can readily be washed out of the membrane or filter under pressure-driven conditions, and then the complex can be dissociated and the desired substance displaced with the effluent in a pure and concentrated state.
Thus, this invention provides new and advantageous ways of effecting separations for analytical and preparative purposes and can be compared with the conventional process of affinity chromatography. Specific advantages of these pressure-driven affinity sorption systems are further set forth in U.S. Pat. No. 4,163,714.